Feature Articles

Pushing the Boundaries in Chromatography

Waters Acquity UPC2 System simplifies laboratory workflow, separates structurally similar compounds, and provides an orthogonal mode of separation to HPLC/UHPLC for full characterization of samples, the company reports.

In 1964, University of Utah Chemistry Professor J. Calvin Giddings enunciated a theoretical platform that would provide liquid chromatography (LC) with resolving power equivalent to that of gas chromatography (GC). “Unified separation science” became a buzzword soon after.

What Giddings envisioned, in the words of Waters scientists Christopher Hudalla and Patrick McDonald, was the “higher mobile phase diffusion and efficiency” of GC, and the “higher selectivity via orthogonal modes of separation” from LC, which could be achieved by combining supercritical carbon dioxide as the major mobile-phase component.

By contrast, conventional high-performance liquid chromatography (HPLC) solvents are toxic and expensive, in both their acquisition and disposal. Aside from water, acetonitrile is arguably the most used solvent in HPLC. It has become prohibitively expensive in recent years; users pay “both ways” for the luxury of using the overwhelming majority of solvents. A four-liter bottle of HPLC-grade acetonitrile costs between $300 and $400. Then, because acetonitrile is generally not recycled due to its high boiling point, disposing of spent mobile-phase diluted with buffers and other organic solvents easily doubles the cost of using the solvent. The carbon footprint for manufacturing acetonitrile is substantial as well, both in terms of atom economy, purification, and shipping.

Supercritical carbon dioxide is the greenest HPLC solvent, hands down. The carbon dioxide is extracted from the atmosphere, to where it is vented after use. Greenhouse gas contribution is zero.

Convergence Chromatography

Waters has named its new-generation supercritical fluid chromatography (SFC) system UltraPerformance Convergence Chromatography, or UPC2 ®.

Convergence chromatography exploits the advantage of carbon dioxide-based mobile phases in either supercritical or subcritical mode. UPC2 combines the unique properties of carbon dioxide mobile phases with the ability to run gradients with common organic solvents, like methanol or acetonitrile, on stationary phases that have both normal- and reverse-phase characteristics, to cover a wide selectivity range.

Like other LC systems built on Waters Acquity® platform, UPC2 employs sub-two-micron stationary phases, especially for nonchiral analysis. It is possible to use five-micron particle technology as well, as with UHPLC, but also like conventional LC, efficiency increases as particle size decreases. Efficiency improvements from smaller particles are in fact comparable to those obtained in HPLC/UHPLC. UPC2’s additional benefit is much higher linear velocity and throughput.

“Mass transfer kinetics are greatly improved. Diffusivity is closer to that of GC’s, between 10 and 100 times faster than that of LC,” Fountain says.

Waters’ original target market was chiral analysis, where SFC already enjoyed a healthy reputation. The company has demonstrated chiral SFC separations that require one-thirtieth the time compared with standard normal-phase HPLC. Solvent consumption and lower cost are additional benefits. In a typical example, the SFC method consumed 135 microliters of methanol, compared with 10 mL of hexane/ethanol for HPLC. Overall cost savings are dramatic, falling from about $6 for a conventional HPLC run to $0.05 for UPC2.

“When you consider this savings on the scale of hundreds to thousands of injections, the financial impact to an organization can be quite exceptional,” says Waters’ John van Antwerp.

Since its selectivity overlaps significantly with normal-phase chromatography, SFC is orthogonal to reverse-phase LC. The technique is applicable to a diverse range of compounds, including most organic-soluble compounds, most salts of organic acids and bases, strong organic acids and bases, small lipophilic peptides, and nonpolar solutes (e.g., waxes and oils). In addition to being the go-to method for chiral separations, SFC also separates positional isomers and diastereomers, and is compatible with most popular detection modes.

Pushback

Waters’ 2004 introduction of UPLC (the company’s branded version of what later came to be generally referred to as UHPLC) was met with some pushback. Users were concerned about the extraordinarily high operating pressures, the cost of new HPLC systems, and method transfer. Eventually, customers recognized UHPLC as an extension of HPLC—a way to exploit the benefits of sub-2 micron stationary phases. The pushback with UPC2 was based on the perception that SFC lacked robustness, was not easy to use, and was less-than ideal outside of chiral separations.

Adapting supercritical chromatography to everyday analytics required more than simply pumping supercritical carbon dioxide through an existing instrument. Fountain makes the analogy to the early development of UPLC. Making minor adjustments to existing columns or instruments while employing 1.7-micron particles with large diameter columns would have been easy. “But we knew from practice and theory that the advantages of sub-two-micron particles would disappear. That was the rationale for redesigning columns, hardware, end fittings, and the instrument itself. That is how the Acquity UPC2 System came to be.”

Waters has gone out of its way to differentiate UPC2 from conventional SFC. Both use carbon dioxide-based mobile phases, and not without justification. “Convergence chromatography brings the SFC practice into mainstream analytical techniques,” Fountain explains. The most formidable hurdle to “instrumenting” SFC in the form of UPC2 was to make the new platform as robust and reliable as traditional analytical chromatography.

“Laboratory scientists know deep down that GC and LC are robust. They trust those technologies. We wanted to bring that same level of trust to SFC,” he says. “We’re not trying to replace GC or LC. But we are finding successful application areas we did not anticipate during development, for example in the areas of semiconductors and organic light-emitting diodes.”

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